Thermal unfolding of G-actin monitored with the DNase I-inhibition assay -: Stabilities of actin isoforms

被引:58
作者
Schüler, H
Lindberg, U
Schutt, CE
Karlsson, R [1 ]
机构
[1] Stockholm Univ, Wenner Gren Inst, Dept Cell Biol, S-10691 Stockholm, Sweden
[2] Princeton Univ, Dept Chem, Henry Hoyt Lab, Princeton, NJ 08544 USA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 02期
关键词
circular dichroism; irreversible denaturation; nucleotide binding; profilin; phosphate analogs;
D O I
10.1046/j.1432-1327.2000.01023.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Actin is one of the proteins that rely on chaperonins for proper folding. This paper shows that the thermal unfolding of G-actin, as studied by GD and ultraviolet difference spectrometry, coincides with a loss in DNase I-inhibiting activity of the protein. Thus, the DNase I inhibition assay should be useful for systematic studies of actin unfolding and refolding. Using this assay, we have investigated how the thermal stability of actin is affected by either Ca2+ or Mg2+ at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in different states of phosphorylation (ATP, ADP.P-i, ADP.V-i, ADP.AlF4, ADP.BeFx, and ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclude that the thermal unfolding of G-actin is a three-state process, in which an equilibrium exists between native actin with bound nucleotide and an intermediate free of nucleotide. Actins in the Mg-form were less stable than the Ca-forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle alpha-actin = bovine cytoplasmic gamma-actin > yeast actin > cytoplasmic p-actin. The activation energies for the thermal unfolding reactions were in the range 200-290 kJ.mol(-1), depending on the bound ligands. Generally, the stability of the actin depended on the degree with which the nucleotide contributed to the connectivity between the two domains of the protein.
引用
收藏
页码:476 / 486
页数:11
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